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GB 31658.11-2021 English PDF

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GB 31658.11-2021: National food safety standard - Determination of albendazole and its metabolites residues in animal derived food by high performance liquid chromatography method
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Basic data

Standard ID GB 31658.11-2021 (GB31658.11-2021)
Description (Translated English) National food safety standard - Determination of albendazole and its metabolites residues in animal derived food by high performance liquid chromatography method
Sector / Industry National Standard
Classification of Chinese Standard X04
Word Count Estimation 8,854
Issuing agency(ies) National Health Commission of the People's Republic of China, State Administration for Market Regulation

GB 31658.11-2021: National food safety standard - Determination of albendazole and its metabolites residues in animal derived food by high performance liquid chromatography method


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National food safety standards Albendazole and its metabolites in animal foods Determination of residues by high performance liquid chromatography National Standards of People's Republic of China Released by the National Health Commission of the People's Republic of China State Administration for Market Regulation Ministry of Agriculture and Rural Affairs of the People's Republic of China

Foreword

This document complies with the provisions of GB/T 1:1-2020 "Standardization Work Guidelines Part 1: Structure and Drafting Rules of Standardization Documents" Drafting: This document is published for the first time: National food safety standards Determination of albendazole and its metabolites residues in animal foods HPLC

1 Scope

This document specifies the sample preparation and HPLC determination methods for the detection of albendazole and its metabolites residues in animal foods: This document applies to albendazole, albendazole sulfone, albendazole sulfoxide, and albendazole-2-aminosulfone in muscles, livers, kidneys, adipose tissues and milk of pigs, cattle, sheep, and chickens Detection of residual amounts:

2 Normative reference documents

The contents of the following documents constitute essential provisions of this document through normative citations in the text: Among them, the cited documents with dates are: Only the version corresponding to the date applies to this document; for undated referenced documents, the latest version (including all amendments) applies to this document: document: GB/T 6682 Specifications and test methods for water used in analytical laboratories

3 Terms and definitions

There are no terms or definitions that need to be defined in this document:

4 Principles

The albendazole and its metabolites remaining in the sample were alkalized and extracted with ethyl acetate (fat sample was extracted with hydrochloric acid water-acetonitrile solution, milk The sample was extracted with acetonitrile and ethyl acetate), purified with acidic alumina solid phase extraction column and mixed cation exchange solid phase extraction column, and high performance liquid phase Detection by chromatography-fluorescence detector and quantification by external standard method:

5 Reagents and materials

Unless otherwise specified, all reagents are of analytical grade and the water is first-grade water that complies with GB/T 6682: 5:1 Reagents 5:2 Solution preparation 5:2:1 2% sodium carbonate solution: Take 2:0g of sodium carbonate, dissolve it in water and dilute it to 100mL: 5:2:2 0:2mol/L hydrochloric acid solution: Take 1:8mL of hydrochloric acid and dilute to 100mL with water: 5:2:3 0:2mol/L hydrochloric acid-acetonitrile solution: Take 10mL of 0:2mol/L hydrochloric acid solution, add 90mL of acetonitrile, and mix: 5:2:4 10mol/L sodium hydroxide solution: Take 4:0g of sodium hydroxide, dissolve and dilute to 10mL with water, and cool to room temperature: 5:2:5 0:004g/mL sodium metabisulfite solution: Take 0:4g of sodium metabisulfite, dissolve it in water and dilute it to 100mL: 5:2:6 0:1mol/L disodium ethylenediaminetetraacetate solution: Take 3:36g of disodium ethylenediaminetetraacetate, dissolve it in water and dilute it to 100mL: 5:2:7 10% ammoniated acetonitrile solution: Take 10 mL of ammonia water, add 90 mL of acetonitrile, and mix well: 5:2:8 0:5mol/L ammonium acetate solution: Take 1:93g ammonium acetate, dissolve it in water and dilute it to 500mL, adjust the pH to 5:0 with glacial acetic acid, and mix well: 5:2:9 Reconstitute the solution: Take 10 mL of acetonitrile and 10 mL of methanol, add 80 mL of 0:5 mol/L ammonium acetate solution, and mix well: 5:2:10 Acetonitrile-dimethyl sulfoxide (9:1): Take 90 mL of acetonitrile and 10 mL of dimethyl sulfoxide, and mix well: 5:3 Standard products 5:3:1 Albendazole (Albendazole, C12H15N3O2S, CAS number: 54965-21-8): content ≥ 99:0%: 5:3:2 Albendazole Sulfone (C12H15N3O4S, CAS number: 75184-71-3): content ≥ 98:3%: 5:3:4 Albendazole-2-aminosulfone (Albendazole-2-aminosulfone, C10H13N3O2S, CAS number: 80983-34-2): content ≥ 90:0%: 5:4 Preparation of standard solutions 5:4:2 Mix standard intermediate solution: accurately pipette 2 mL of albendazole standard stock solution, 0:2 mL of albendazole sulfone standard stock solution, 2 mL of albendazole sulfoxide standard stock solution and albendazole respectively -2-Aminosulfone standard stock solution 1mL, in a 10mL volumetric flask, dilute to the mark with acetonitrile, prepare albendazole concentration of 20μg/mL, albendazole sulfone concentration of 2μg/mL, albendazole sulfone concentration Mixed standard intermediate solution with a sulfone concentration of 20 μg/mL and albendazole-2-aminosulfone concentration of 10 μg/mL: Ready to use: 5:5 Materials 5:5:1 Acidic alumina solid phase extraction column: 1g/3mL, the filler is acidic alumina, or equivalent: 5:5:2 Mixed cation exchange solid phase extraction column: 150mg/6mL, or equivalent: 5:5:3 Nylon microporous filter membrane: 0:22μm:

6 Instruments and equipment

6:1 High performance liquid chromatograph: equipped with fluorescence detector: 6:2 Analytical balance: sensitivity 0:01g and sensitivity 0:00001g: 6:3 Tissue homogenizer: 6:4 Vortex mixer: 6:5 Vortex oscillator: 6:6 Centrifuge: the speed can reach 10000r/min: 6:7 Solid phase extraction device: 6:8 Nitrogen blower: 7: Preparation and preservation of samples 7:1 Preparation of samples 7:1:1 Organization Take an appropriate amount of fresh or thawed blank or test tissue, mince it, and homogenize it: a) Take a homogeneous test sample as a test material; b) Take a homogeneous blank sample as a blank sample; c) Take a homogeneous blank sample, add a standard solution of appropriate concentration, and add the sample as a blank: 7:1:2 Milk Take an appropriate amount of fresh or thawed blank or test milk sample and mix evenly: a) Take a homogeneous test sample as a test material; b) Take a homogeneous blank sample as a blank sample; c) Take a homogeneous blank sample, add a standard solution of appropriate concentration, and add the sample as a blank: 7:2 Storage of samples Store below -18℃:

8 Measurement steps

8:1 Extraction 8:1:1 Muscle, liver, kidney Take 2g of the sample (accurate to ±0:02g), place it in a 50mL centrifuge tube, add 0:5mL of 2% sodium carbonate solution, 1mL of dimethyl sulfoxide, Add 1 mL of 0:004 g/mL sodium metabisulfite solution, vortex for 1 min: Add 10 mL of ethyl acetate, vortex for 1 min, shake for 5 min, and centrifuge at 10000 r/min: Incubate for 5 minutes and take the supernatant: Add 10 mL of ethyl acetate to the residue and repeat the extraction once: Combine the supernatants and blow with nitrogen at 40°C until nearly dry: Add the residue to normal Add 5 mL of hexane, vortex for 1 min, then add 2 mL of 0:2 mol/L hydrochloric acid-acetonitrile solution, vortex for 1 min, let stand and separate into layers, discard the upper layer of n-hexane: Remove the lower solution and set aside: 8:1:2 Fat Take 2g of the sample (accurate to ±0:02g), place it in a 50mL centrifuge tube, heat it in a 70°C water bath to melt it, and add 2% sodium carbonate solution 0:5 mL, 0:2 mol/L hydrochloric acid water-acetonitrile solution 5 mL, vortex for 1 min, shake for 5 min, centrifuge at 10000 r/min for 5 min, take the supernatant: Heat the residue in a 70°C water bath to melt it, add 0:2 mol/L Repeat the extraction of 5 mL of hydrochloric acid water-acetonitrile solution, and combine the supernatants: Add 5 mL of n-hexane, vortex for 1 min, let stand for layering, discard the upper layer of n-hexane, add 5 mL of n-hexane, repeat the operation, and remove the layer solution for later use: 8:1:3 Milk Take 2g of the sample (accurate to ±0:02g), place it in a 50mL centrifuge tube, add 50μL of 10mol/L sodium hydroxide solution, 0:5mL of 0:004g/mL sodium metabisulfite solution, vortex for 1 minute, add 10mL of acetonitrile, Add 5 mL of ethyl acetate, vortex, shake for 5 min, centrifuge at 10,000 r/min for 5 min, and remove the supernatant: Add 5 g of anhydrous sodium sulfate, shake for 5 min, centrifuge at 10,000 r/min for 5 min, remove the supernatant, and blow with nitrogen at 40°C until nearly dry: :Add 5 mL of n-hexane to the residue, vortex for 1 min, then add 2 mL of 0:2 mol/L hydrochloric acid water-acetonitrile solution, vortex for 1 min, let stand for layering, discard the upper layer of n-hexane, and remove the layer solution for later use: 8:2 Purification The acidic alumina solid phase extraction column was dissolved in 3 mL of 0:2 mol/L hydrochloric acid water-acetonitrile solution and 0:1 mol/L disodium ethylenediaminetetraacetate: Activate with 3 mL of solution, pass the reserve solution through the column, collect the effluent, elute with 1 mL of 0:2 mol/L hydrochloric acid-acetonitrile solution, combine the effluents, and add Add 8 mL of 0:2 mol/L hydrochloric acid solution and mix well: Then add 3 mL of methanol, 3 mL of 0:1 mol/L disodium ethylenediaminetetraacetate solution, and water in sequence: 3 mL, 3 mL of 0:2 mol/L hydrochloric acid solution activated mixed cation exchange solid phase extraction column, elute with 3 mL of 0:2 mol/L hydrochloric acid solution, 3 mL of methanol, drain for 1 min, and elute with 5 mL of 10% ammoniated acetonitrile solution: Drain, collect the eluate, and blow dry with nitrogen in a 40°C water bath: Dissolve the residue in 1:0 mL of solution and pass it through a nylon microporous filter for high performance liquid chromatography measurement (the solution on the machine should be measured within 72 hours): 8:3 Preparation of standard curve Accurately pipette an appropriate amount of the mixed standard intermediate solution, and dilute albendazole and albendazole sulfoxide with the complex solution to a concentration of 0:05 μg/mL: 0:1 μg/mL, 0:2 μg/mL, 0:5 μg/mL, 1 μg/mL, 2 μg/mL, albendazole sulfone concentration is 0:005 μg/mL, 0:01 μg/mL, 0:02 μg/mL, 0:05 μg/mL, 0:1 μg/mL, 0:2 μg/mL, albendazole-2-aminosulfone concentration is 0:025 μg/mL, 0:05 μg/mL, A series of standard working solutions of 0:1μg/mL, 0:2μg/mL, 0:5μg/mL, and 1μg/mL are used for high-performance liquid chromatography determination: Taking the peak area of the test substance as the ordinate, the corresponding standard solution concentration is On the abscissa, draw the standard curve: Find the regression equation and correlation coefficient: 8:4 Determination 8:4:1 Liquid chromatography reference conditions a) Chromatographic column: C18 (150mm×4:6mm, 5μm), or equivalent; b) Column temperature: 30℃; c) Injection volume: 20μL; d) Excitation wavelength: 290nm; e) Emission wavelength: 330nm; f) Mobile phase: A is acetonitrile, B is methanol, and C is 0:5 mol/L ammonium acetate solution: The gradient elution conditions are shown in Table 1: 8:4:2 Determination method Take the sample solution and the corresponding standard solution, perform single-point or multi-point calibration, and quantify the chromatographic peak area according to the external standard method: Standard working solution and sample The response values of albendazole and its metabolites in the solution should be within the linear range of instrument detection: The peak retention of albendazole and its metabolites in the sample The relative deviation between the time and the retention time of the corresponding peak of the standard working solution should be within ±2:5%: Chromatogram of the standard solution of albendazole and its metabolites See Appendix A: 8:5 Blank test Take a blank sample and perform parallel operations using the same measurement steps except that no standard solution is added:

9 Calculation and presentation of results

The residual amount of the substance to be tested in the sample is calculated according to the standard curve or formula (1): 10 Sensitivity, accuracy and precision of detection methods 10:1 Sensitivity This method detects albendazole, albendazole sulfone, albendazole sulfoxide, and albendazole-2-aminosulfone in the muscles, livers, kidneys, adipose tissues and milk of pigs, cattle, sheep, and chickens The limits are all 0:02 mg/kg, and the limits of quantitation are all 0:05 mg/kg: 10:2 Accuracy This method uses albendazole, albendazole sulfone, albendazole sulfoxide, and albendazole-2-aminosulfone in the muscles, livers, kidneys, adipose tissues and milk of pigs, cattle, sheep, and chickens: The recovery rate at the added concentration of 05mg/kg~10mg/kg is 60%~120%: 10:3 Precision The intra-batch relative standard deviation of this method is ≤15%, and the inter-batch relative standard deviation is ≤20%:

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